Hiroko Bannai

Spatiotemporal calcium dynamics in single astrocytes and its modulation by neuronal activity

Astrocytes produce a complex repertoire of Ca2+ events that coordinate their major functions. The principle of Ca2+ events integration in astrocytes, however, is unknown. Here we analyze whole Ca2+ events, which were defined as spatiotemporally interconnected transient Ca2+ increases. Using such analysis in single hippocampal astrocytes in culture and in slices we found that spreads and durations of Ca2+ events follow power law distributions, a fingerprint of scale-free systems. A mathematical model demonstrated that such Ca2+ dynamics can arise from intracellular inositol-3-phosphate diffusion. The power law exponent (α) was decreased by activation of metabotropic glutamate receptors (mGluRs) either by specific receptor agonist or by low frequency stimulation of glutamatergic fibers in hippocampal slices. Decrease in α indicated an increase in proportion of large Ca2+ events. Notably, mGluRs activation did not increase the frequency of whole Ca2+ events. This result suggests that neuronal activity does not trigger new Ca2+ events in astrocytes (detectable by our methods), but modulates the properties of existing ones. Thus, our results provide a new perspective on how astrocyte responds to neuronal activity by changing its Ca2+ dynamics, which might further affect local network by triggering release of gliotransmitters and by modulating local blood flow.

Astrocytic IP3Rs: Contribution to Ca2+signalling and hippocampal LTP: Astrocytic IP3Rs: Ca2+Signalling and LTP

Astrocytes regulate hippocampal synaptic plasticity by the Ca2+ dependent release of the N‐methyl d‐aspartate receptor (NMDAR) co‐agonist d‐serine. Previous evidence indicated that d‐serine release would be regulated by the intracellular Ca2+ release channel IP3 receptor (IP3R), however, genetic deletion of IP3R2, the putative astrocytic IP3R subtype, had no impact on synaptic plasticity or transmission. Although IP3R2 is widely believed to be the only functional IP3R in astrocytes, three IP3R subtypes (1, 2, and 3) have been identified in vertebrates. Therefore, to better understand gliotransmission, we investigated the functionality of IP3R and the contribution of the three IP3R subtypes to Ca2+ signalling. As a proxy for gliotransmission, we found that long‐term potentiation (LTP) was impaired by dialyzing astrocytes with the broad IP3R blocker heparin, and rescued by exogenous d‐serine, indicating that astrocytic IP3Rs regulate d‐serine release. To explore which IP3R subtypes are functional in astrocytes, we used pharmacology and two‐photon Ca2+ imaging of hippocampal slices from transgenic mice (IP3R2−/− and IP3R2−/−;3−/−). This approach revealed that underneath IP3R2‐mediated global Ca2+ events are an overlooked class of IP3R‐mediated local events, occurring in astroglial processes. Notably, multiple IP3Rs were recruited by high frequency stimulation of the Schaffer collaterals, a classical LTP induction protocol. Together, these findings show the dependence of LTP and gliotransmission on Ca2+ release by astrocytic IP3Rs. GLIA 2017;65:502–513

Astrocytic IP3Rs: Contribution to Ca2+ signalling and hippocampal LTP

Astrocytes regulate hippocampal synaptic plasticity by the Ca2+ dependent release of the N‐methyl d‐aspartate receptor (NMDAR) co‐agonist d‐serine. Previous evidence indicated that d‐serine release would be regulated by the intracellular Ca2+ release channel IP3 receptor (IP3R), however, genetic deletion of IP3R2, the putative astrocytic IP3R subtype, had no impact on synaptic plasticity or transmission. Although IP3R2 is widely believed to be the only functional IP3R in astrocytes, three IP3R subtypes (1, 2, and 3) have been identified in vertebrates. Therefore, to better understand gliotransmission, we investigated the functionality of IP3R and the contribution of the three IP3R subtypes to Ca2+ signalling. As a proxy for gliotransmission, we found that long‐term potentiation (LTP) was impaired by dialyzing astrocytes with the broad IP3R blocker heparin, and rescued by exogenous d‐serine, indicating that astrocytic IP3Rs regulate d‐serine release. To explore which IP3R subtypes are functional in astrocytes, we used pharmacology and two‐photon Ca2+ imaging of hippocampal slices from transgenic mice (IP3R2−/− and IP3R2−/−;3−/−). This approach revealed that underneath IP3R2‐mediated global Ca2+ events are an overlooked class of IP3R‐mediated local events, occurring in astroglial processes. Notably, multiple IP3Rs were recruited by high frequency stimulation of the Schaffer collaterals, a classical LTP induction protocol. Together, these findings show the dependence of LTP and gliotransmission on Ca2+ release by astrocytic IP3Rs. GLIA 2017;65:502–513

Basal ryanodine receptor activity suppresses autophagic flux

Graphical abstract Figure. No Caption available. ABSTRACT The inositol 1,4,5‐trisphosphate receptors (IP3Rs) and intracellular Ca2+ signaling are critically involved in regulating different steps of autophagy, a lysosomal degradation pathway. The ryanodine receptors (RyR), intracellular Ca2+‐release channels mainly expressed in excitable cell types including muscle and neurons, have however not yet been extensively studied in relation to autophagy. Yet, aberrant expression and excessive activity of RyRs in these tissues has been implicated in the onset of several diseases including Alzheimer’s disease, where impaired autophagy regulation contributes to the pathology. In this study, we determined whether pharmacological RyR inhibition could modulate autophagic flux in ectopic RyR‐expressing models, like HEK293 cells and in cell types that endogenously express RyRs, like C2C12 myoblasts and primary hippocampal neurons. Importantly, RyR3 overexpression in HEK293 cells impaired the autophagic flux. Conversely, in all cell models tested, pharmacological inhibition of endogenous or ectopically expressed RyRs, using dantrolene or ryanodine, augmented autophagic flux by increasing lysosomal turn‐over (number of autophagosomes and autolysosomes measured as mCherry‐LC3 punctae/cell increased from 70.37 ± 7.81 in control HEK RyR3 cells to 111.18 ± 7.72 and 98.14 ± 7.31 after dantrolene and ryanodine treatments, respectively). Moreover, in differentiated C2C12 cells, transmission electron microscopy demonstrated that dantrolene treatment decreased the number of early autophagic vacuoles from 5.9 ± 2.97 to 1.8 ± 1.03 per cellular cross section. The modulation of the autophagic flux could be linked to the functional inhibition of RyR channels as both RyR inhibitors efficiently diminished the number of cells showing spontaneous RyR3 activity in the HEK293 cell model (from 41.14% ± 2.12 in control cells to 18.70% ± 2.25 and 9.74% ± 2.67 after dantrolene and ryanodine treatments, respectively). In conclusion, basal RyR‐mediated Ca2+‐release events suppress autophagic flux at the level of the lysosomes.

Imaging mGluR5 Dynamics in Astrocytes Using Quantum Dots

This unit describes the method that we have developed to clarify endogenous mGluR5 (etabotropic tamate eceptors 5) dynamics in astrocytes by single‐particle tracking using quantum dots (QD‐SPT). QD‐SPT has been a powerful tool to examine the contribution of neurotransmitter receptor dynamics to synaptic plasticity. Neurotransmitter receptors are also expressed in astrocytes, the most abundant form of glial cell in the brain. mGluR5s, which evoke intracellular Ca2+ signals upon receiving glutamate, contribute to the modulation of synaptic transmission efficacy and local blood flow by astrocytes. QD‐SPT has previously revealed that the regulation of the lateral diffusion of mGluR5 on the plasma membrane is important for local Ca2+ signaling in astrocytes. Determining how mGluR5 dynamics are regulated in response to neuronal input would enable a better understanding of neuron‐astrocyte communication in future studies. Curr. Protoc. Neurosci. 66:2.21.1‐2.21.18.

Astroglial Ca 2+ signaling is generated by the coordination of IP 3 R and store-operated Ca 2+ channels

Astrocytes play key roles in the central nervous system and regulate local blood flow and synaptic transmission via intracellular calcium (Ca2+) signaling. Astrocytic Ca2+ signals are generated by multiple pathways: Ca2+ release from the endoplasmic reticulum (ER) via the inositol 1, 4, 5-trisphosphate receptor (IP3R) and Ca2+ influx through various Ca2+ channels on the plasma membrane. However, the Ca2+ channels involved in astrocytic Ca2+ homeostasis or signaling have not been fully characterized. Here, we demonstrate that spontaneous astrocytic Ca2+ transients in cultured hippocampal astrocytes were induced by cooperation between the Ca2+ release from the ER and the Ca2+ influx through store-operated calcium channels (SOCCs) on the plasma membrane. Ca2+ imaging with plasma membrane targeted GCaMP6f revealed that spontaneous astroglial Ca2+ transients were impaired by pharmacological blockade of not only Ca2+ release through IP3Rs, but also Ca2+ influx through SOCCs. Loss of SOCC activity resulted in the depletion of ER Ca2+, suggesting that SOCCs are activated without store depletion in hippocampal astrocytes. Our findings indicate that sustained SOCC activity, together with that of the sarco-endoplasmic reticulum Ca2+-ATPase, contribute to the maintenance of astrocytic Ca2+ store levels, ultimately enabling astrocytic Ca2+ signaling.

Optimal microscopic systems for long-term imaging of intracellular calcium using a ratiometric genetically-encoded calcium indicator.

Monitoring the pattern of intracellular Ca(2+) signals that control many diverse cellular processes is essential for understanding regulatory mechanisms of cellular functions. Various genetically encoded Ca(2+) indicators (GECIs) are used for monitoring intracellular Ca(2+) changes under several types of microscope systems. However, it has not yet been explored which microscopic system is ideal for long-term imaging of the spatiotemporal patterns of Ca(2+) signals using GECIs. We here compared the Ca(2+) signals reported by a fluorescence resonance energy transfer (FRET)-based ratiometric GECI, yellow cameleon 3.60 (YC3.60), stably expressed in DT40 B lymphocytes, using three different imaging systems. These systems included a wide-field fluorescent microscope, a multipoint scanning confocal system, and a single-point scanning confocal system. The degree of photobleaching and the signal-to-noise ratio of YC3.60 in DT40 cells were highly dependent on the fluorescence excitation method, although the total illumination energy was maintained at a constant level within each of the imaging systems. More strikingly, the Ca(2+) responses evoked by B-cell antigen receptor stimulation in YC3.60-expressing DT40 cells were different among the imaging systems, and markedly affected by the illumination power used. Our results suggest that optimization of the imaging system, including illumination and acquisition conditions, is crucial for accurate visualization of intracellular Ca(2+) signals.

Astrocytic IP3 Rs: Contribution to Ca(2+) signalling and hippocampal LTP.

Astrocytes regulate hippocampal synaptic plasticity by the Ca2+ dependent release of the N‐methyl d‐aspartate receptor (NMDAR) co‐agonist d‐serine. Previous evidence indicated that d‐serine release would be regulated by the intracellular Ca2+ release channel IP3 receptor (IP3R), however, genetic deletion of IP3R2, the putative astrocytic IP3R subtype, had no impact on synaptic plasticity or transmission. Although IP3R2 is widely believed to be the only functional IP3R in astrocytes, three IP3R subtypes (1, 2, and 3) have been identified in vertebrates. Therefore, to better understand gliotransmission, we investigated the functionality of IP3R and the contribution of the three IP3R subtypes to Ca2+ signalling. As a proxy for gliotransmission, we found that long‐term potentiation (LTP) was impaired by dialyzing astrocytes with the broad IP3R blocker heparin, and rescued by exogenous d‐serine, indicating that astrocytic IP3Rs regulate d‐serine release. To explore which IP3R subtypes are functional in astrocytes, we used pharmacology and two‐photon Ca2+ imaging of hippocampal slices from transgenic mice (IP3R2−/− and IP3R2−/−;3−/−). This approach revealed that underneath IP3R2‐mediated global Ca2+ events are an overlooked class of IP3R‐mediated local events, occurring in astroglial processes. Notably, multiple IP3Rs were recruited by high frequency stimulation of the Schaffer collaterals, a classical LTP induction protocol. Together, these findings show the dependence of LTP and gliotransmission on Ca2+ release by astrocytic IP3Rs. GLIA 2017;65:502–513